This invention relates to an apparatus and a method for optimizing the separation of a liquid chromatographic sample and, more specifically, to an apparatus and method for optimizing such separation with respect to an optimization parameter.
In the analytical method of liquid chromatography, a sample to be analyzed is injected together with a suitable solvent (mobile phase) into a separation column, typically under very high pressure. The various components of the sample interact differently with the column material and the solvent such that the components elute from the column at different times. The substances leaving the column are subsequently detected by a detector such as, for example, a photometric detector. The time between the injection of a sample and the detection of a specific component is called the retention time for that component. The results of chromatographic separations are displayed as a plot of detector signal versus time, commonly known in the art as a chromatogram. A chromatogram typically comprises a plurality of peaks, wherein each peak corresponds to a certain component of the sample to be analyzed. The area of the peak is to some degree characteristic of the amount of the respective component present in the sample.
In order to ensure a reliable qualitative and quantitative analysis of the sample, it is necessary that the chromatogram has a good overall resolution, which is characterized by distinct, narrow peaks for different components. In chromatograms with bad overall resolution, it is frequently not possible to so recognize that adjacent peaks actually correspond to different sample components. Likewise, it may not be possible to derive meaningful quantitative values for the amounts of these components present. It is known that the overall resolution of a chromatogram for a specific sample depends upon the composition of the mobile phase and that an improvement of this resolution can be achieved by varying this composition as, for example, by blending several solvents in a certain ratio.
Thus, much effort has been directed to the development of mobile phase compositions resulting in optimal liquid chromatographic separations. One method for the selection of the optimum mobile phase composition in liquid chromatography is known from A. C. J. H. Drouen, et al., "An Improved Optimization Procedure for the Selection of Mixed Mobile Phases in Reversed Phase Liquid Chromatography", Cromatorapia, Vol. 16, pages 48-52, which is incorporated herein by reference. This method is an iterative optimization approach wherein some chromatograms of a sample to be examined are measured at different solvent compositions and, based upon the measured chromatograms, a so-called retention model is calculated which describes the chromatographic capacity factor as a function of solvent composition for the various components of the sample. Using this retention model, an overall resolution value is derived for each possible solvent composition, whereby the overall resolution value is a measure of the quality of the chromatographic separation for the respective solvent compositions. The chromatographer then initiates a new chromatographic separation with a solvent composition having a resolution value at or near the maximum of the optimal separation. The new experimental data thus obtained are used to refine the retention model, from which a new resolution value function is derived. The procedure can be repeated until the optimum mobile phase has been found.
However, this known optimization procedures is not satisfactory in all respects. Depending upon the definition of the overall resolution value, the optimization procedure may lead to different results, that is, to different solvent compositions which are regarded as optimal. Furthermore, the resolution value may not be sufficiently discriminating between different chromatographic separations; the resolution values associated with different separations are identical or very close to each other so that it is difficult to predict where optimal conditions are to be searched. One way to overcome these difficulties is to define overall resolution in a way which will provide better discrimination. However, even such redefined resolution is not satisfactory for all separation problems. Consequently, differing optimization criteria are sometimes employed, leading to a complication of the optimization procedure. Furthermore, this known method only offers limited flexibility. If, for example, the chromatographer is interested in an optimal separation of one of the sample's components, the known method, which is based on determining an overall resolution, is of limited use.